Our goal is to design targeted therapies for autoimmune thyroid diseases (AITD) by dissecting the mechanisms by which HLA class II molecules interact with thyroidal peptides to induce AITD. In the last grant cycle, we made substantial progress toward these goals: (1) We have shown that the presence of arginine at position beta 74 of the HLA-DR peptide binding pocket (DRb1-Arg74) is key to the development of AITD; (2) We identified 4 thyroglobulin (Tg) peptides that bind strongly to the DRb1-Arg74 pocket. One of the peptides (Tg.2098), is a major T-cellepitope;(3) We identified a small molecule and a D-peptide that block the binding of Tg.2098 to DRb1-Arg74; (4) We discovered epigenetic interactions between interferon & Tg mediated via IRF-1; (5) We (& others) showed that TSHR is a major Graves' disease (GD) gene that has synergism with DRb1-Arg74 in conferring risk for GD. This synergism suggests that, similar to Tg, there is interaction between TSHR peptides & DRb1-Arg74 pockets. We propose building on these findings to identify blockers of DRb1-Arg74 as potential new therapies for AITD. Our hypothesis is that binding of pathogenic thyroidal peptides to the DRb1-Arg74 pocket & their presentation to T-cells are critical to the etiology of AITD. Blocking the binding of peptides to MHC II may be used to treat AITD. Specific Aims: Specific Aim 1: To identify pathogenic TSH receptor (TSHR) peptides that bind to DRb1-Arg74. We will use: (1) Molecular modeling: to predict which of the > 750 potential TSHR peptides can bind to DRb1- Arg74 pockets; (2) Biochemical and Mass Spectrometry studies to confirm peptides that bind to DRb1-Arg74; (3) T-cell studies to test whether peptide binders can stimulate T-cells from a GD mouse model and from GD patients. Specific Aim 2: To screen for small molecule inhibitors (SMI's) of peptide - DRb1-Arg74 binding. We will use: (1) Computer screen of a large library of compounds (~ 115,000) to identify potential SMI's that bind to DRb1-Arg74 pockets; (2) High throughput experimental screening (HTS) of a library of ~ 115,000 compounds; (3) Confirmation of lead compounds by in vitro binding inhibition assays using recombinant DRb1-Arg74, and by inhibition of T-cell activation by Tg/TSHR peptides. Specific Aim 3: Identifying & analyzing D-amino acid peptide blockers of peptide-DRb1-Arg74 binding. We will use: (1) In silico screen of D-peptides that are predicted to block the DRb1-Arg74 pocket to peptide binding; (2) In vitro confirmation of predicted D-peptides by inhibition assays of binding to recombinant DRb1-Arg74; (3) T-cell assays to test whether predicted D-peptide blockers can inhibit proliferative responses of lymphocytes isolated from mice with auto immune thyroid it is and from patients with AITD. In summary, this multidisciplinary translational project builds directly on the knowledge gained in the previous grant period. Our goals are to develop novel treatment modalities for AITD using SMI and D-peptides to block peptide HCIIbinding. Our collaborative team has the capacity, experience, & expertise to achieve our aims. The main advantage of our approach is that it is actually capable of achieving the rapeutic specificity since only peptides that bind to Arg74+ pockets will be blocked. Our translational studies will hopefully lead to novel therapies for AITD.